The purposes of the studies are to develop a transtracheally-inoculated primate model of Pneumocystis carinii (P. carinii f.sp.simiae) that will allow studies which require a reproducible severe infection model and to use that model to test compounds and study organisms in air samples to determine possible transmission factors. The primate model will be very important for determining therapeutic and prophylactic effects of drugs. Currently rodents are used to predict drug efficacy for humans and it is known that some drugs (and their enantiomers) are metabolized or activated differently in rodents than in primates. It is known that rhesus macaques are susceptible to Pneumocystis spp because outbreaks of infection have occurred in SIV-infected animals. The outbreaks have been spontaneous and a controlled model of infection is needed as well as a sensitive method for detection of infection and a typing method to identify strains of Pneumocystis sp found in animals and air samples. Initial studies focused on study of organisms from infected lungs banked at Tulane to work out the best primers and methods for polymerase chain reaction (PCR) amplification of nuclear or mitochondrial rRNA. Studies of small portions of the 18S and 26S rRNA genes, the entire 5.8 rRNA gene and the internal spacers ITS1 and ITS2 of one specimen of simian Pneumocystis were cloned and sequenced. A portion of the large subunit mitochondrial rRNA gene was also cloned and sequenced. There were marked differences in the ITS1 and ITS2 and mitochonrial gene sequences of the simian P. carinii as compared to the same sequences of other hosts. These studies establshed the methods to amplify the specific gene portions, establish a distinct type of P. carinii from this monkey and demonstrate the capability of detecting and typing organisms from other macaques. In order to determine whether the types detected are specific within macaques or whether the types found are some of a range, requires study of many more lung samples. The sequence variabilities found have been reported in an article submitted to the Journal of Clinical Microbiology. Thirteen specimens from Tulane currently are being studied. To date sequences have been established for 3 of these. In the others, cloning is being carried on. Plasmids with inserts are being selected by picking of colonies with appropriate characteristics. The work will require several more weeks before comparisons of sequences will be available. Once types found in the thirteen lung samples are determined, type or types in the specimen used for inoculation can be established and this type or these ypes quantitated in the lungs of inoculated animals. The next step in development of the model is use of an immunosuppressive agent in established doses that will deplete lymphocytes by 50% or greater and allow inoculation of P. carinii and subsequent development of infections. Dr. Bohm is developing the immunosuppression doses and we are prepared to help in a trial inoculation procedure for two animals. Testing for the development of infections will require our collaboration on sampling the animals by bronchoscopy (performed by Dr. Bohm) with histochemical staining (by Marilyn Bartlett) and PCR amplification (performed by Dr. Lee). With establishment of the model, we can schedule both drug treatment trials and air sampling experiments.